Optimization of capillary electrophoresis conditions for a glucagon competitive immunoassay using response surface methodology

The capillary electrophoresis (CE) conditions for a competitive immunoassay of glucagon were optimized for highest sensitivity of the immunoassay and resolution of the electrophoretic peaks using a Box-Behnken design. Injection time, voltage ramp time, and separation voltage were varied between three levels and two responses, bound-to-free (B/F) ratio of the immunoassay peaks and resolution between the peaks, were measured. Analysis of variance was applied to fit a predictive model, and a desirability function was used to simultaneously optimize both responses. A 10-s injection, 1.6-min ramp time, and a 22-kV separation voltage were the conditions found when high B/F was given more emphasis than high resolution. To test the model, calibration curves of a glucagon immunoassay were measured at the optimum and least optimum CE conditions. Optimal conditions increased the sensitivity of the immunoassay by 388% compared to the least optimum conditions while maintaining adequate resolution.